Glucuronidation of Fatty Acids by Human ER & Nuclear UGT

人内质网对脂肪酸的葡萄糖醛酸化

• 批准号: 6645333
• 负责人: Anna Radominska-Pandya
• 金额: $ 27.51万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-15 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6645333
• 关键词: Glucuronidation; Fatty; Acids; Human; ER

DESCRIPTION (Provided by applicant): Fatty acids (FAs) are important structural components of cell membranes, energy sources and precursors of eicosanoids. FAs can also act as second messengers and regulators of signal transduction. By these mechanisms, FAs play a significant physiological role in controlling cellular events such as growth, differentiation, proliferation and apoptosis. It has been documented that oxidized fatty acids (OFAs), including eicosanoids, are biosynthesized and excreted in the form of glucuronides in pathological conditions in humans. The physiological function of glucuronidation of free fatty acids (FFAs) is less developed. However, the in vivo biosynthesis of glucuronides of FFAs has been confirmed by the identification of carboxyl-linked glucuronides of FFAs in human urine. As a major objective of this proposal, we will characterize FA glucuronidation in endoplasmic reticulum (ER) and nuclear membranes in human tissues. The central hypothesis for this project is that FAs and their oxidixed derivatives are physiologically important substrates for human UGTs and glucuronidation plays a protective role against elevated levels of certain FAs. This research proposal will focus on the determination of the catalytic and molecular properties of UDP-glucuronosyltransferases (UGTs) of the UGT2B subfamily that glucuronidate OFAs and FFAs in humans. Specific Aims 1 and 2 will characterize the catalytic properties of UGTs involved in FA glucuronidation. We will identify FAs that are substrates for human UGT isoforms. Substrate specificity and substrate-inhibitor interactions will be investigated. We will biosynthesize FA glucuronides and study their potential toxicity and their effect on the expression of UGTs in tissue cultures. In Specific Aims 3 and 4, structure-function relationship studies will be performed. The structural domains of UGTs required for effective glucuronidation will be studied. Amino acid motifs localized in substrate binding sites or required for cellular targeting to the ER and nuclear membranes will be identified. Photoaffinity labeling studies with photoactive FAs will be carried out to identify amino acids involved in FA binding. Targeting of UGTs to ER and nuclear membranes will be investigated using green fluorescent protein fusions and immunofluorescence studies. Site-directed mutagenesis studies will confirm the importance of structural domains. The information obtained from our studies on FA glucuronidation will provide not only a rationale for understanding FA detoxification in response to elevated levels of various FAs but also information which can be used as important therapeutic strategies, such as the development of drugs targeting cardiovascular disease, inflammatory responses and cancer.

描述（由申请人提供）：脂肪酸 (FA) 是重要的结构 细胞膜的成分、能源和类二十烷酸的前体。 FA 还可以充当第二信使和信号转导的调节者。经过 在这些机制中，FAs 在控制中发挥着重要的生理作用。 细胞事件，例如生长、分化、增殖和凋亡。 据记载，氧化脂肪酸（OFA），包括类二十烷酸， 在病理过程中以葡萄糖醛酸苷的形式生物合成和排泄 人类的条件。游离葡萄糖醛酸化的生理功能 脂肪酸（FFA）尚不发达。然而，体内生物合成 FFA 的葡萄糖苷酸已通过鉴定得到证实 人尿液中 FFA 的羧基连接的葡萄糖醛酸苷。 作为本提案的主要目标，我们将表征 FA 葡萄糖醛酸化 存在于人体组织的内质网 (ER) 和核膜中。这 该项目的中心假设是 FA 及其氧化衍生物 是人类 UGT 和葡萄糖醛酸化的重要生理底物 对某些 FA 水平升高具有保护作用。这项研究 该提案将重点关注催化和分子的确定 UGT2B 亚家族的 UDP-葡萄糖醛酸基转移酶 (UGT) 的特性 人类体内的葡萄糖醛酸 OFA 和 FFA。具体目标 1 和 2 将描述 UGT 参与 FA 葡萄糖醛酸化的催化特性。我们将 识别作为人类 UGT 同工型底物的 FA。底物特异性 并将研究底物-抑制剂相互作用。我们将 生物合成 FA 葡萄糖醛酸苷并研究其潜在毒性及其 对组织培养中 UGT 表达的影响。在具体目标 3 和 4 中， 将进行结构-功能关系研究。结构性 将研究有效葡萄糖醛酸化所需的 UGT 域。氨基 酸性基序位于底物结合位点或细胞所需 将确定针对 ER 和核膜的靶向。光亲和力 将进行光敏 FA 标记研究，以鉴定氨基 参与 FA 结合的酸。 UGT 靶向 ER 和核膜 将使用绿色荧光蛋白融合物进行研究 免疫荧光研究。定点诱变研究将证实 结构域的重要性。 我们从 FA 葡萄糖醛酸化研究中获得的信息将提供 不仅是理解 FA 解毒的基本原理 各种 FA 水平升高，而且还有可用作 重要的治疗策略，例如靶向药物的开发 心血管疾病、炎症反应和癌症。